Isolation of Staphylococcus pseudintermedius in Immunocompromised Patients from a Single Center in Spain: A Zoonotic Pathogen from Companion Animals

Staphylococcus pseudintermedius, a commensal opportunistic bacterium predominantly residing in the skin of companion animals, particularly dogs, has the potential to induce skin and soft tissue infections in pets, and zoonotic infections, including catheter-related complications. This study documents four cases of S. pseudintermedius infection or colonization in patients who had close contact with dogs or cats. Identification of the bacterial species was performed using MALDI-TOF mass spectrometry, and antibiotic susceptibility was determined using microdilution assay. DNA was sequenced using Nanopore technology followed by in silico analysis. Three isolates were multidrug resistant, including resistance to methicillin, with one belonging to the prevalent European lineage ST551, and the other two were attributed to a novel multilocus sequence type, ST2672. The remaining isolate was attributed to the novel multilocus sequence type ST2673 and was methicillin susceptible. All four isolates exhibited an array of virulence factors that contributed to colonization, damage to host immune cells, and biofilm formation. All the ST551 isolates included in the comparative analysis displayed clonality within the European continent. The importance of describing zoonotic infections associated with S. pseudintermedius resides in the scarcity of available scientific literature, further accentuated by its heightened resistance profile and potential complications, particularly in the context of catheter-related infections.


Introduction
Over the years, there has been a tendency to misclassify coagulase-positive (CoPS) Staphylococcus isolates, with most being identified as Staphylococcus aureus.In 1976, Hájek identified a novel CoPS, Staphylococcus intermedius, from pigeons, dogs, minks, and horses, noting its distinctive biochemical properties [1].Over the following years, various Staphylococcus species were added to the S. intermedius group (SIG), including Staphylococcus pseudintermedius in 2005 [2].Despite recognizing S. pseudintermedius as a species, classification challenges persist due to similar biochemical properties [3].
S. pseudintermedius is a commensal opportunistic bacterium found in approximately 90% of dogs and also in other companion animals like cats, inhabiting skin and mucocutaneous sites such as the nose, mouth, groin, and perineum [4,5].The transition from commensal to opportunistic behavior may be influenced by skin lesions, compromised immunity, antibiotic treatment, and external factors, often leading to skin and soft tissue infections, otitis externa, urinary tract infections, and postoperative infections [4,6,7].Notably, S. pseudintermedius often harbors methicillin resistance (MRSP), mainly due to the mecA gene within the Staphylococcus chromosome cassette mec (SCCmec) [8][9][10].MRSP strains typically exhibit multidrug resistance (MDR) [11], posing treatment challenges and raising concerns about zoonotic potential [12].
A widely used method for classifying and establishing clonal associations of S. pseudintermedius is multilocus sequence typing (MLST).In Europe, the predominant sequence type is ST71, which exhibits high antibiotic resistance.However, other MLST are gaining relevance, such as ST496 in France, known for having resistance to a broad range of antibiotics, as well as ST258 and ST551 [4,9,[13][14][15][16].
Unlike in dogs, colonization by S. pseudintermedius in humans is usually sporadic or considered as contamination [17][18][19][20][21], with immunocompromised patients [22,23] and veterinary dermatologists [24] being the most likely to be colonized by this microorganism.Even though a mortality rate for S. pseudintermedius infections in humans has not yet been determined, there have been fatal cases involving this organism.Balkhed et al. described a case of a 77-year-old male with fatal bacteremia, most likely caused by S. pseudintermedius, possibly transmitted by one of the dogs with which he shared a household [25].In their 2023 publication, Moses et al. [3] summarized reports of S. pseudintermedius colonization and infection in humans from 2006 to 2022, including 48 studies with only three from Spain [26][27][28].This study describes four cases of S. pseudintermedius infections in immunocompromised patients who had been in contact with dogs and/or cats.It provides a comprehensive analysis of these strains, evaluating their antibiotic resistance profiles both phenotypically and genotypically, and assessing the presence of virulence factors.

Patients and Isolates
From February to June 2023, four individuals with diverse comorbidities attended at the Hospital Clínic de Barcelona and S. pseudintermedius was isolated from skin (infection or colonization).

DNA Extraction and Sequencing
DNA extraction was conducted using the ZymoBIOMICS TM DNA miniprep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer's protocol.Subsequently, the assessment of DNA quality and quantity was performed using the NanoDrop TM 2000 Spectrophotometer and Quantus TM Fluorometer with the QuantiFluor ® dsDNA System (Promega, Madison, WI, USA), respectively.Oxford Nanopore Technologies (ONT) libraries were prepared with approximately 400 ng of DNA using the Rapid Barcoding Sequencing kit (SQK-RBK004, ONT, Oxford, UK).Sequencing was carried out on a MinION FLO-MIN106 v9.1.4flow cell (ONT, Oxford, UK) and the MinION Mk1C device (ONT, Oxford, UK).Live basecalling was chosen, and reads with a quality score < 8 and ≤200 bp were excluded from further analysis.
MLST were determined using PubMLST [33] and MLST 2.0 from the Center for Genomic Epidemiology (CGE) [34].Three isolates presented two previously undescribed sequence types (23-298 and 23-583 shared the same new allele combination).The sevenallele combination for these isolates was submitted to the S. pseudintermedius PubMLST typing database, and a novel sequence type was assigned to both combinations.
Since isolate 23-150 belonged to the MLST ST551, a search for ST551 genomes was conducted within the NCBI Nucleotide database using the query "pseudintermedius AND ST551", from which we retrieved 19 genomes (1 from Felis catus, 13 from Canis lupus familiaris, and 5 from environmental samples).Representative genomes corresponding to ST159 and ST498, the nearest sequence types to the newly identified ST2673 in this study, were also sought, retrieving one and two genomes isolated from dogs, respectively.The terms "pseudintermedius and "Homo sapiens"" were concurrently used to retrieve the first 23 genomes of S. pseudintermedius isolated from human cases.Consequently, our dataset encompassed a total of 50 genomes, including the reference genome (CP065921.1)and the isolates from our study (refer to Supplementary Table S1 for details of all the genomes from NCBI).Phylogeny was inferred by calling single nucleotide polymorphisms (SNPs) using CSIPhylogeny [35] v1.4.The Newick file obtained was visualized using FigTree v1.4.4 (available at http://tree.bio.ed.ac.uk/software/figtree/, accessed on 15 January 2024).Average nucleotide identity (ANI) against the 46 downloaded genomes was calculated with FastANI [36].

Medical History and Isolates
Retrospectively, all skin and soft tissue samples with S. pseudintermedius isolated from January to July 2023 in the Clinical Microbiology department of the Hospital Clínic de Barcelona were included (Table 1).

Antibiotic Susceptibility Profile
Susceptibility testing (Table 2) showed that ST511 (23-150) and ST2672 (23-298 and 23-583) isolates were MRSP and MDR, resistant to at least six antibiotic families: β-lactams, macrolides, lincosamides, fluoroquinolones, trimethoprim-sulfamethoxazole, and tetracyclines.Isolate 23-150 was only susceptible to vancomycin, rifampicin, and linezolid, while ST2672 isolates were susceptible to gentamicin, vancomycin, rifampicin, and linezolid.Isolate 23-472 (ST2673) was susceptible to all tested antibiotics.Table 2. Minimum inhibitory concentrations (MICs, µg/mL) and antibiotic resistance genotype.All the strains that presented the blaZ gene also presented the blaI and blaR1 genes from the same operon.Shaded boxes denote resistance in the case of antibiotics, and presence in the case of antibiotic resistance genes.Supplementary Table S2 shows antibiotic susceptibility from additional studies of ST551 isolates.While not all studies examined identical antibiotics, concordant antibiotics showed similar susceptibility patterns to isolate 23-150.

Antibiotic Resistance Genotype
In silico analysis identified 11 ARGs, and two and one mutations in the gyrA and parC genes, respectively (Table 2).
Isolate 23-150 (ST551) had more ARGs than the ST2672 isolates.It contained the blaZ gene, the mecA gene within an SCCmec type Vc, the aac(6 ′ )-Ie-aph(2 ′′ )-Ia gene, the erm(B) gene, the dfrG gene, and the tet(M) gene.It lacked the tet(L) gene but had the tet(K) gene.Additionally, it had the aph(3 ′ )-IIIa and ant(6)-Ia genes for aminoglycoside resistance and the sat4 gene for streptothricin resistance.Compared to other ST551 isolates (Supplementary Table S3), they showed a nearly identical ARG profile; only one isolate lacked ant(6)-Ia and erm(B).

Antibiotic Resistance Genes' Genomic Context
The genomic context of all ARGs, excluding blaZ, was examined.Figure 1a shows a distinct cluster of five genes (aph(3′)-IIIa, sat4, ant(6)-Ia, erm(B), and dfrG) in isolate 23-150.The first three genes are flanked by insertion sequence elements.In contrast, ST2672 isolates (23-298 and 23-583) lack these three genes, with erm(B) adjacent to two tRNA genes.For tetracycline resistance (Figure 1b), isolate 23-150 had both tet(K) and tet(M) genes.The tet(K) gene was located between plasmid-related genes, suggesting possible plasmid integration into the chromosome.The tet(M) gene was surrounded by elements that might aid in its mobilization.In ST2672 isolates, the tet(L) and tet(M) genes were adjacent, indicating potential plasmid integration of the tet(L) gene.
The aac(6 ′ )-Ie-aph(2 ′′ )-Ia gene was found in isolate 23-150 and ST2672 isolates (Figure 1c), sharing a similar genomic context, differing only by the presence of a "group II intron reverse transcriptase/maturase" element.Downstream of this gene, genes encoding phage elements were annotated.
The basic structure of SCCmec includes three main components: the ccr gene complex (coding for recombinases that mobilize the cassette); the mec gene complex (harboring the mecA gene, regulator genes, and insertion sequences like IS431); and a J region containing non-essential elements.Isolate 23-150 had SCCmec V(5C2) according to SCCmecFinder, while isolates 23-298 and 23-583 had a pseudocassette lacking the ccr gene complex, denoted as ΨSCCmec 57395 (Figure 2a).
The aac(6′)-Ie-aph(2″)-Ia gene was found in isolate 23-150 and ST2672 isolates (Figure 1c), sharing a similar genomic context, differing only by the presence of a "group II intron reverse transcriptase/maturase" element.Downstream of this gene, genes encoding phage elements were annotated.
The basic structure of SCCmec includes three main components: the ccr gene complex (coding for recombinases that mobilize the cassette); the mec gene complex (harboring the mecA gene, regulator genes, and insertion sequences like IS431); and a J region containing non-essential elements.Isolate 23-150 had SCCmec V(5C2) according to SCCmecFinder, while isolates 23-298 and 23-583 had a pseudocassette lacking the ccr gene complex, denoted as ΨSCCmec57395 (Figure 2a).In red, mecA gene; in orange, elements responsible for mobilization and reorganization of the cassette; in light blue, ∆mecR1; in green, gray, and teal, genes responsible for arsenic, cadmium, and copper resistance, respectively.Homology gradients range from green (100%) to yellow (85%), and violet to light blue (85%) for inverted regions.
The SCCmec V(5C2) in isolate 23-150 showed 100% homology with the SCCmec V(5C2) sequence from S. aureus JCSC3624 (WIS) (AB121219.1)(Figure 2a, first and second lines).It included two copies of IS431 in opposite directions flanking the mec gene complex, with one copy truncating the mecR1 regulatory gene, and two copies of the recombinase ccrC.In contrast, isolates 23-298 and 23-583 lacked the ccr gene complex, with the mecA gene and one copy of IS431 inverted (Figure 2a, second and third lines).
Isolates 23-298 and 23-583 had a genomic configuration for the mecA gene lacking the ccr gene complex, showing 100% homology with the ΨSCCmec 57395 element identified in S. pseudintermedius strain 57395 by Perreten et al. [45], except for the absence of an IS256 tnp gene in our isolates (Figure 2a, third and fourth lines).This pseudocassette includes two IS431 copies in the same orientation, as well as resistance genes for cadmium (cadC and cadX), arsenic (arsB, arsC, and arsR), and copper (copA), situated adjacent to the IS431 copies.A similar configuration with metal-resistance genes has been observed in other staphylococci, such as Staphylococcus haemolyticus, as described by Zong [46] (Figure 2a, fifth line).Additionally, other SCCmec types have been reported to contain the same metalresistance genes but in a different configuration, like in SCCmec IX(1C2), where all the genes are clustered (Figure 2a, sixth line).
An analysis of 46 genomes from NCBI revealed 52 VF genes, with several unique genes compared to the study's isolates.Notably, genes encoding surface proteins (spsF, spsO, spsP, and spsQ) and toxins (exi, expB, and seccanine) were present in a minority of the genomes, indicating variability in virulence factors among different strains.

Phylogenetic and ANI Analysis
Phylogenetic analysis based on SNPs and ANI analysis compared the isolates' genomes with genomes from NCBI, including ST551 genomes and other human isolates (Figure 3).All ST551 genomes from NCBI were of canine origin, except for one from a cat, and were distributed across countries like Spain, Italy, Norway, and Switzerland.
Isolate 23-150 (ST551) showed high ANI identity with other ST551 isolates, ranging from 99.79% to 99.9%.All ST551 genomes formed a distinct cluster within the phylogenetic tree, with isolate 23-150 closely related to isolate 18-H48227, sharing a 99.9% ANI.Isolates 23-298 and 23-583 showed the highest identity similarity with a ST45 isolate from Thailand (VB88), both in ANI identity and phylogenetic placement.Isolate 23-472 had its highest ANI similarity with an ST1518 isolate from the UK (17-23) but clustered more closely with isolate MRSP8510 ST1412 from Argentina in the phylogenetic tree, both from human cases.The four study isolates had the least homology with the ST150 isolate from the USA, with ANI ranging from 98.40% to 98.66%.pseudintermedius genomes available at NCBI nucleotide database, and other S. pseudintermedius isolated from Homo sapiens.ANI results are expressed as percentages and displayed in a heat map, with green boxes indicating the highest identities and red boxes indicating the lowest identities.Information regarding host disease, isolation source, and sequencing technology used can be found in Supplementary Table S1.MLST, multilocus sequence type; UK, United Kingdom; USA, United States of America.

Discussion
S. pseudintermedius is part of the canine skin microbiota and can cause zoonotic infections.Historically, it has been misidentified as other Staphylococcus species, such as S. aureus and S. intermedius [51], due to, for example, limitations in API databases lacking comprehensive representation of zoonotic pathogens.Moreover, the delayed and poor response of S. pseudintermedius to slide coagulase and commercial latex agglutination tests has led to its erroneous categorization as coagulase-negative Staphylococcus [3].The introduction of MALDI-TOF has significantly reduced these misidentifications [52].
We identified an ST551 isolate from a 79-year-old man with frequent contact with his son's dog.This strain showed an MDR MRSP profile, resistant to 8 of 11 antibiotics tested (Table 2), and containing 10 ARGs, plus mutations in 2 fluoroquinolone resistance genes (Table 2).Comparative analysis with other ST551 isolates from various countries (Supplementary Tables S2 and S3) revealed a consistent resistance pattern, both genotypically and phenotypically.The genetic context of the ARGs (Figure 1) aligns with previous descriptions by Viñes et al. [11], featuring a five-gene cluster (aph(3 ′ )-IIIa, sat4, ant(6)-Ia, erm(B), dfrG), and mecA within an SCCmec type Vc element.Additionally, tet(K) was found in a putative plasmid inserted into the chromosome, consistent with previous findings.All ST551 isolates analyzed shared a nearly identical virulence factor profile, with only three genes (spsD, spsI, and spsL) differing (Supplementary Table S4).Isolate 23-150 included these three genes, with SpsD and SpsL being known to play roles in S. pseudintermedius infection [49,65].The consistent ANI values and phylogenetic relatedness (Figure 3) suggest the ST551 lineage is clonal and has spread across Europe with minor genetic changes.
Two of the S. pseudintermedius isolates (23-298 and 23-583) were recovered from peritoneal dialysis pericatheter infection and colonization in two young women (33 and 36 years old, respectively).Both isolates belonged to ST2672, exhibiting identical antibiotic resistance patterns and virulence factor profiles.Notably, both patients owned companion animals, specifically two dogs and a cat, respectively.The association between S. pseudintermedius infections and catheters has been previously documented.In 2010, Chuang et al. [66] described a catheter-related bacteremia in a 6-year-old boy, initially misidentified as S. intermedius but later confirmed as S. pseudintermedius through 16S rRNA analysis.The boy had been exposed to a companion dog.In 2019, Diaz et al. [67] reported a catheter-related bloodstream infection in a 36-year-old woman undergoing hemodialysis who had one cat and four dogs.A subsequent case study by Nomoto et al. [68] detailed a 41-year-old man with a central parenteral nutrition system featuring a totally implantable venous access port (TIVAP).Despite being advised to avoid contact with his pet dog, the patient experienced a reinfection 52 days after discharge, with the strain matching the initial one.After avoiding contact with the dog, no further reinfection occurred.
Hence, the potential acquisition of an identical ST2672 S. pseudintermedius clone by both patients from their respective companion animals remains plausible.Bierowiec et al. [63] reported a 22.86% MRSP prevalence in cats, Papić et al. [15] identified one ST71 MRSP strain from a cat's wound, Van Duijkeren et al. [69] isolated MRSP strains from cystitis and otitis externa in two cats, and Abraham et al. [70] reported methicillin-resistant S. intermedius (presumably S. pseudintermedius) in a cat in 2007.However, the concurrence of the same clone in unrelated individuals suggests a potential nosocomial infection source within the dialysis unit, rather than two separate community-acquired infections through animal contact.To elucidate origin of the strain, comprehensive investigations, including environmental samples from the hospital and patients' pets, are needed.Such inquiries are essential to determine whether the hospital environment serves as a reservoir for S. pseudintermedius.
All isolates examined in this study harbored several virulence factor genes crucial for S. pseudintermedius pathogenesis, including colonization, biofilm formation, and induction of host inflammatory reactions.As Kmieciak et al. noted [71], virulence factors such as the elastin-binding protein Ebps (ebpS gene), fibrinogen/fibronectin binding protein SpsE (spsE gene), and the exfoliative toxin SIET (siet gene) contribute to superficial infections.Toxins like leukocidin (lukF-I and lukS-I genes) can damage host defense cells and erythrocyte membranes, compromising function and aiding colonization spread [72].
Genes critical for effective colonization, particularly in catheter-related infections, include those mediating biofilm formation.All four isolates harbored the icaABCD operon and the psmαβδε genes.The icaABCD operon is involved in synthesizing the polysaccharide intercellular adhesin, while the psmαβδε genes produce modulins that damage host cells and stabilize biofilms.Pompilio et al. [73] found that S. pseudintermedius strains associated with human wounds can generate ultrastructurally complex biofilms, providing a protective environment against antibiotics and enhancing pathogenic potential.This underscores the ability of these strains to cause implant-associated infections, such as the catheter-related infections that represented 50% of the cases in this study, posing a risk of life-threatening complications.
To prevent or reduce zoonotic transmissions of S. pseudintermedius from dogs to humans, pet owners should be aware of behaviors that could potentially lead to transmission, including feeding dogs in the kitchen, allowing dogs to sleep in the kitchen or on the sofa, and letting dogs lick their faces and/or hands [74], as hands seem to play an important role in transmission via contaminated surfaces [75].Additionally, S. pseudintermedius can persist in the environment for up to 10 weeks [18].Regarding the issue of MDR isolates, better antibiotic treatment practices should be considered.Mandatory susceptibility testing has been shown to reduce antimicrobial resistance, as does avoiding the misuse of antibiotics through empirical treatment rather than treatment based on susceptibility testing [24,76,77].

Conclusions
In conclusion, this study documents four cases of human S. pseudintermedius infection or colonization in individuals in contact with companion animals.Two clonally related isolates were recovered from unrelated women with peritoneal dialysis catheters, and another isolate belonged to the increasingly prevalent ST551 MLST in Europe.Three out of the four isolates exhibited a MDR genotype and phenotype, and all demonstrated a diverse array of virulence factors linked to the microorganism's pathogenicity.The scarcity of reported S. pseudintermedius human cases, particularly in Spain, underscores the need for more studies on its role in zoonotic infections.Given the widespread ownership of dogs, natural reservoirs of this microorganism, the resistance of different S. pseudintermedius lineages to multiple antibiotics poses a potential threat to infection treatment.To determine if the hospital environment acts as a reservoir for S. pseudintermedius, thorough investigations, including environmental samples from the hospital and patients' pets, are needed.

Figure 2 .
Figure 2. Genomic context of mecA.(a) SnapGene Viewer visualization of the genomic context of mecA in isolates 23-150 and 23-298.(b) EasyFig comparison of the genomic context of mecA in isolates

Figure 3 .
Figure 3. Phylogenetic tree and ANI.We performed phylogenetic analysis with all the ST551 S. pseudintermedius genomes available at NCBI nucleotide database, and other S. pseudintermedius isolated from Homo sapiens.ANI results are expressed as percentages and displayed in a heat map, with

Figure 3 .
Figure3.Phylogenetic tree and ANI.We performed phylogenetic analysis with all the ST551 S. pseudintermedius genomes available at NCBI nucleotide database, and other S. pseudintermedius isolated from Homo sapiens.ANI results are expressed as percentages and displayed in a heat map, with green boxes indicating the highest identities and red boxes indicating the lowest identities.Information regarding host disease, isolation source, and sequencing technology used can be found in Supplementary TableS1.MLST, multilocus sequence type; UK, United Kingdom; USA, United States of America.

Table 1 .
Information of the host and isolates included in this study.These are all the S. pseudintermedius collected in daily routine in the Microbiology department of the Hospital Clínic de Barcelona from February to June (both included) of 2023.